Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CAHoward Hughes Medical Institute, Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA

Department of Molecular and Cellular Biology, School of Biological Sciences, University of Adelaide, Adelaide, South Australia, AustraliaCentre for Molecular Pathology, School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia

Introduction

Differentiation of activated B cells during the initial stages of T cell–dependent antibody responses proceeds simultaneously along pathways leading to early (extrafollicular) plasmablasts (PBs), germinal center (GC) B cells, and GC-independent, early memory B cells. These pathways differ in their spatiotemporal emergence, the longevity of their end products, their affinity for antigens, and their functional capacity (Taylor et al., 2012) and are considered important for establishing robust and diverse antibody responses. Adoption of these fates is controlled in part by B cell–trafficking receptors, which are dynamically regulated after antigen engagement to enable B cell access to antigens, interactions with T cells, and positioning in distinct lymphoid niches that foster the formation of immediate or long-lasting, antigen-specific antibody responses (Pereira et al., 2010). How antigen-activated B cells regulate their response to the several chemoattractants to which they may be simultaneously or sequentially exposed is uncertain. It is, however, potentially crucial as a mechanism in determining stoichiometry in the distribution of B cells along the differentiation pathways that generate the effector B cells of the immune response.

Results and discussion

Although a previous study (Heinzel et al., 2007) concluded that ACKR4 is expressed exclusively by cells of nonhematopoietic origin in unimmunized mice, we detected ACKR4 transcripts and protein expression by GC B cells (Fig. 1, A and B). To investigate the possible functions for hematopoietic ACKR4 in T cell–dependent humoral immunity, we used bone marrow (BM) chimerism to generate mice in which ACKR4 deficiency was restricted to the hematopoietic compartment (H-Ackr4−/−). We immunized these H-Ackr4−/− mice with sheep red blood cells (SRBCs) and observed an increased frequency of GC B cells at all time points assessed after immunization but most prominently on day 5 relative to hematopoietic WT (H-WT) mice (Fig. 1 C). The number of T follicular helper (TFH) cells, mediators of GC B cell selection and proliferation, and T follicular regulatory (TFR) cells, implicated in regulating the magnitude of the GC reaction, were also increased in immunized H-Ackr4−/− mice relative to controls (Fig. S1 A). The formation of early PBs, despite a lack of detectable ACKR4 expression, in H-Ackr4−/− mice was also enhanced on day 5 of the response (Fig. 1 D). These data reveal a negative regulatory role for ACKR4 expression in the hematopoietic compartment on early PB and GC B cell development. To determine whether this effect was intrinsic to B cell expression of ACKR4, we reconstituted lethally irradiated mice with a 4:1 mixture of BM recovered from B cell–deficient (μMT) mice that lack endogenous B cells (Kitamura et al., 1991) and either WT (B-WT) or Ackr4−/− (B-Ackr4−/−) mice. Immunizing B-Ackr4−/− mice with SRBC reproduced the increased numbers of splenic GC B cells, TFH cells, and early PBs seen in immunized H-Ackr4−/− mice (Fig. 1, E and F; and Fig. S1 B). Collectively, these data reveal a B cell–intrinsic regulatory role for ACKR4 in the early stages of the B cell response to a T cell–dependent antigen.

Our findings in H-Ackr4−/− and B-Ackr4−/− mice indicated that ACKR4 negatively regulates both early PB and GC B cell responses (Fig. 1, C–G). Recent evidence has indicated that a single B cell clone can enter all three possible differentiation fates, and that this positively correlates with the magnitude of their early proliferation and resistance to apoptosis (Taylor et al., 2015). Based on these observations and our data, we hypothesized that ACKR4 functions in undifferentiated, activated B cells to negatively regulate both early PB and GC B cell responses. This hypothesis predicts that Ackr4-deficient B cells will preferentially enter early PB and GC B compartments in an environment in which Ackr4−/− and WT B cells are in competition. To test this, we studied Ackr4−/− early PB and early GC B cell responses (day 5 after SRBC administration) in the context of mixed BM chimeras. In mice reconstituted with an equal mixture of Ackr4−/− and WT BM, ACKR4 deficiency enhanced early PB and early GC B cell responses relative to concurrently activated WT cells (Fig. 3 A). To extend these observations, we studied concurrent WT (CD45.1/2) and Ackr4−/− (CD45.2) SWHEL B cell responses to the reduced affinity HEL mutant HEL2X (Ka = 8 × 107 M−1). This response forms early PBs, GC B cells, and a population of cells (B220+Ig[HEL]hiGL7-) that likely encompass early memory B cells (Chan et al., 2009; Brink et al., 2015) by day 5 after immunization. At this time point, Ackr4−/− SWHEL B cells outcompeted WT SWHEL B cells in the early PB and GC B and B220+HELhiGL7− cell populations, most prominently among SWHEL early PBs (Fig. 3 B). We hypothesized further that enforced expression of ACKR4 prior to B cell activation by antigens may limit entry into early PB and GC B cell compartments. We tested this by generating Ackr4 knockin mice (Rosa26LSL-Ackr4; Fig. S2 A) and making expression conditional to follicular (Fo) B cells by introducing a Cre recombinase driven by the regulatory elements of Cd23 (Cd23Cre; Kwon et al., 2008). Fo B cells from Cd23Cre/+.Rosa26LSL-Ackr4/+ mice expressed functional ACKR4 (Fig. S2, B and C) and had frequencies of splenic Fo B cells and marginal-zone B cells that were equivalent to WT littermates (Fig. S2 D). Transgenic ACKR4 expression by Fo B cells did not alter CCR7 or CXCR5 expression at rest or after anti–IgM stimulation (Fig. S2 E), but, consistent with the known function of ACKR4 as a scavenger of CCR7 ligands (Comerford et al., 2006, 2010), inhibited anti-IgM–stimulated B cell migration toward CCL21, but not CXCL13, a non-ACKR4 ligand in mice (Townson and Nibbs, 2002; Fig. S2 F). Supporting the hypothesis that ACKR4 negatively regulates activated B cell differentiation, in mixed BM chimeric mice reconstituted with Cd23Cre/+.Rosa26LSL‑Ackr4/+ and WT BM, ACKR4 transgenic B cells were less represented among early PB and GC B cells that form on day 5 of the SRBC response (Fig. 3 C). These data suggest that enforced ACKR4 expression in Fo B cells limits their ability to form early PB and GC B cells when in competition with WT cells. Collectively, we conclude that ACKR4 negatively regulates early PB and GC B cell responses in a B cell–intrinsic manner. Furthermore, these experiments did not reveal a cell-intrinsic role for ACKR4 in TFH or TFR development (Fig. S1 C), suggesting that the enhanced T cell responses observed in H-Ackr4−/− and B-Ackr4−/− mice (Fig. S1, A and B) are secondary to enhanced B cell responses, an observation that is in keeping with recent evidence, indicating that the magnitude of the TFH cell response is proportional to the magnitude of the GC B cell response (Baumjohann et al., 2013).

To examine ACKR4-dependent regulation of the initial antigen-engaged B cell differentiation in more detail, we tracked early WT and Ackr4−/− SWHEL B cell responses to HEL2X using mixed SWHEL B cell transfers. Profiling CFSE dilution revealed a cell-intrinsic proliferative advantage for Ackr4−/− over WT SWHEL B cells as early as day 2 (Fig. 4 A), which was increasingly apparent as the response progressed (Fig. 4 B). Notably, the magnitude of the advantage for Ackr4−/− SWHEL B cells on day 3 of the reaction (KO:WT = 1.26 ± 0.01) was similar to their advantage among the day 5 GC B cell (KO:WT = 1.36 ± 0.01; Fig. 3 B) and B220+HELhiGL7− cell (KO:WT = 1.49 ± 0.02; Fig. 3 B) populations. These findings suggest that enhanced early proliferation of responding Ackr4−/− SWHEL B cells may contribute to their accumulation among the effector cell compartments by day 5 of the reaction (Fig. 3 B). In experiments in which WT or Ackr4−/− SWHEL B cells were transferred into separate recipients, Ackr4−/− SWHEL B cells were detected at greater frequencies by day 2.5, and remained more abundant during the first 5.5 d of the response (Fig. 4 C). By day 5.5, mice receiving Ackr4−/− SWHEL B cells had an increased total number of early PBs, GC B cells, and B220+HELhiGL7− cells per spleen compared with controls (Fig. 4 D). Ackr4−/− SWHEL B cells showed a greater propensity to form early PBs (Fig. 4, E and F), which was apparent by flow cytometry on day 4.5 (Fig. 4 G) and translated to increased circulating anti–HEL IgM and IgG1 titers and kinetics (Fig. 4 H). Together, these findings suggest that ACKR4 limits early antigen-engaged B cell proliferation in a B cell–intrinsic manner, reducing the number of antigen-engaged B cell precursors available for early PB and GC B cell differentiation.

To explore the relationship between ACKR4 function as a regulator of CCR7-dependent cellular migration and its negative regulation of activated B cell responses, we studied the distribution of WT and Ackr4−/− SWHEL B cells within the spleen after HEL2X-SRBC immunization using histology (Fig. 5 A). WT and Ackr4−/− SWHEL B cells were located throughout B cell follicles before immunization (unpublished data). Within 24 h, a proportion of WT SWHEL B cells had redistributed along the T/B border or had emigrated into the T cell zone. By days 2–2.5, most of the WT SWHEL B cells remained at the T/B border, although some redistribution to the interfollicular zone (IFZ; defined here as the lateral poles of the Fo B cell proximal to bridging regions, also referred to as the marginal-zone bridging channel) was apparent. In contrast, an increased proportion of Ackr4−/− SWHEL B cells were localized in the IFZ early during the response. On days 2–2.5, a larger proportion of Ackr4−/− SWHEL B cells were localized in the IFZ and were visibly more abundant than the WT SWHEL B cell response at that time, which became increasingly apparent during the next 60 h of the response. Whereas most WT SWHEL B cells were positioned in the IFZ and at the T/B border on days 3–3.5, a proportion of Ackr4−/− SWHEL B cells were distributed in the outer follicle, with the emergence of SWHEL B cell clusters in the outer follicular regions and the IFZ and the appearance of cells exhibiting a PB phenotype in the bridging channels on day 3, which became more obvious by days 3.5–4.5. These early ACKR4-dependent changes to the migratory patterns of SWHEL B cells were independent of detectable cell-intrinsic defects in CCR7, CXCR5, CXCR4, or EBI2 expression during the first 3 d of this response (Fig. 5 B). Thus, in the absence of ACKR4, a proportion of activated B cells preferentially home to the splenic IFZ during the early stages of the humoral immune response. Favorable positioning to interfollicular niches was accompanied with the enhanced expansion of Ackr4−/− SWHEL B cells in these zones and exaggerated early PB responses.

CCR7 guides activated B cell homing toward the T/B border but also contributes to the lateral spreading along that interface and positioning within the splenic IFZ (Reif et al., 2002; Okada et al., 2005; Gatto et al., 2011). To determine whether ACKR4-mediated regulation of early B cell responses was dependent on CCL19 and/or CCL21, these ligands were neutralized in mixed BM chimeras. This revealed that the advantage of ACKR4-deficient B cells to enter early PB and GC B cell compartments was dependent, at least in part, on physiological CCL19/CCL21 (Fig. 5 C).

Concluding remarks

Our findings establish ACKR4 as a B cell–intrinsic regulator of early PB and GC B cell responses. First, using ACKR4-deficient anti-HEL monoclonal B cells, we demonstrate that ACKR4 limits the early migration of antigen-engaged B cells to splenic interfollicular niches. Unrestricted access of activated B cells to these niches in the absence of ACKR4 was associated with their enhanced early expansion, which we propose increased the precursor pool of activated B cells available for subsequent differentiation into early PB and GC B cell fates. Further, we demonstrate that aberrant, splenic IFZ localization by antigen-engaged ACKR4-deficient anti-HEL B cells was accompanied by the preferential formation of early PB responses.

Existing evidence indicates that migration of activated B cells is predominantly shaped by their balanced responsiveness to CCR7, EBI2, CXCR5, and CXCR4 ligands (Pereira et al., 2010). Gatto et al. (2011) demonstrated that transfer of Cxcr5-deficient, activated B cells results in their exclusion from B cell follicles and accumulation in marginal-zone bridging channels; however, compound deletion of Cxcr5 with Ccr7 was shown to direct cells away from this niche and toward the outer regions of the follicle. These data indicate that, in addition to its established role as driving activated B cell migration toward the T cell zone (Reif et al., 2002), CCR7 also contributes to positioning activated B cells toward the IFZ and bridging zones of the spleen. Our experiments with SWHEL B cells indicate that deletion of ACKR4, a scavenger of CCR7 ligands, promotes responding B cell migration to splenic IFZ. We speculate that ACKR4 may function to “tune” early CCR7-dependent cues on a proportion of responding B cells, limiting their CCR7-driven homing to splenic IFZ. Our findings that physiological CCL19 and CCL21 were required, at least in part, for ACKR4-dependent changes to early PB and GC B cell responses supports a relationship between ACKR4 and CCR7 function in the regulation of activated B cell responses.

Our results indicate that the propensity of ACKR4-deficient SWHEL B cells to form early PB responses correlated with their favorable accumulation in the IFZ during the early stages of T cell–dependent humoral immunity. These data, together with published findings that (a) EBI2-deficient B cells, which are defective in their ability to access splenic IFZ and bridging channels, fail to form robust early PB responses (Gatto et al., 2009); (b) forced EBI2 expression on B cells promotes early PB responses (Gatto et al., 2009); and (c) targeted antigen delivery to splenic DCIR2+ dendritic cells, which localize to this niche, elicit robust early PB responses, and are implicated in promoting PB survival (García De Vinuesa et al., 1999; Chappell et al., 2012), support a model in which B cell migration at the early stages of activation has an important role in coordinating and balancing differentiation to early PB and GC B cell fates.

In summary, our results describe an in vivo, cell-intrinsic role for ACKR4 in shaping activated B cell differentiation and further our understanding of the cellular events that govern antibody production.

Materials and methods

Mice

All mice were on the C57BL/6J background and housed in specific pathogen-free conditions at Laboratory Animal Services, University of Adelaide (unless indicated otherwise). C57BL/6 (WT) and B6.Ly5.1 (B6.SJL Ptprca) were purchased from the Animal Resource Center and bred in house. Ackr4−/−, Ccr7−/−, μMT, Ackr4−/−.Ccr7−/− SWHEL (CD45.2), SWHEL (CD45.1/2), and SWHEL.Ackr4−/− (CD45.2) mice were bred and maintained in house. Cd23Cre mice were provided by M. Busslinger (Research Institute of Molecular Pathology, Vienna, Austria). Cd23Cre.Rosa26LSL-Ackr4/+ mice were generated by interbreeding and maintained in house. Mice used in experiments were gender- and age-matched animals and were between the ages of 6 and 12 wk. All animal experiments were approved by the Animal Ethics Committee of the University of Adelaide.

BM chimeras

Recipient mice were lethally irradiated with 1,000 rad (two doses of 500 rad) and reconstituted with 4–5 × 106 total BM cells i.v. of genotypes indicated in text. A minimum of 8 wk was allowed for reconstitution before experimentation.

Histology

For assessment of SWHEL B cell responses by histology (days 1–2.5), 5 × 105 HEL-binding B cells from SWHEL or SWHEL.Ackr4−/− mice were transferred i.v. into WT mice, which were immunized with 2 × 109 HEL2X-SRBC i.v. the next day. For assessment of SWHEL B cell responses by histology (days 3–5.5), 2 × 105 HEL-binding B cells from SWHEL or SWHEL.Ackr4−/− mice were transferred i.v. into WT mice, which were immunized with 109 HEL2X-SRBC i.v. the next day. Organs were frozen in Tissue-Tek optimal cutting temperature–embedding medium (Sakura Finetek). Cryostat sections (8 µm) were fixed in ice-cold acetone and stained, as previously described (Bunting et al., 2013). For detection of HEL-binding B cells, sections were first blocked with 30% normal horse serum and incubated with HELWT (100 ng/ml; Sigma-Aldrich), which was detected using unconjugated rabbit anti–HEL (polyclonal; Rockland) and goat anti–rabbit Ig-Alexa Fluor 488 (Life Technologies). For SWHEL histology, antibodies to IgD (11-26c; eBioscience) and CD4 (RM4-5; BD) were used. For the GC stain (Fig. 2 A), antibodies to IgD, CD3 (145-2C11; BD), BCL-6 (K112-91; BD), and CCL21 (goat polyclonal; R&D) were used. To enumerate transferred SWHEL B cell positioning in spleen sections, the outer follicle, follicle center, T/B interface, IFZs, and T cell zone per white pulp region were first defined (Gatto et al., 2011) in a blinded manner on images stained with IgD/CD4, with HEL-binding fluorescence removed. HEL-binding fluorescence was then merged with these images, and the total number of HEL-binding cells per white pulp area and HEL-binding cells in defined regions were enumerated by four independent researchers. Means ± SEM values across those four independent data sets are presented.

In vivo CCL19/CCL21 neutralization

Affinity-purified anti–mouse (m) CCL21 was generated and purified in house as described (Comerford et al., 2010). Anti–mCCL19 antibodies were raised in New Zealand white rabbits by immunization with full-length, synthetically manufactured CCL19, which was active in calcium mobilization and chemotaxis assays (Clark-Lewis et al., 1994). Serum IgG was purified from preimmunized bleeds (normal rabbit IgG [NRIgG]) and mCCL19- or mCCL21-immunized rabbits using Protein A columns (Millipore). The CCL19- or CCL21-neutralizing ability of these antibodies was confirmed in chemotaxis assays before their use in vivo. Recipient, mixed-BM chimeric mice were administered 500 µg affinity-purified rabbit anti–mCCL21 and 500 µg affinity-purified, rabbit anti–mCCL19 or 1 mg NRIgG i.p. on days −1, 0, 2, and 4. Mice were immunized with SRBCs i.p. on day 0 and analyzed 5 d later.

VH gene sequencing analysis of NP+IgG1+ GC B cells

Single NP+IgG1+ GC B cells were sorted from NP-KLH–immunized (day 14) mice using a BD FACSAria cell sorter. Two rounds of PCR were performed on cDNA using a single proximal 5′ primer for the J558 VH gene family (Ehlich et al., 1994; Smith et al., 2000), together with nested primers specific for Cγ1 (McHeyzer-Williams et al., 1991; Smith et al., 2000). Bands of expected size were purified, sequenced, and analyzed for VH186.2-containing sequences as described. For VH186.2+ clones, the region encoding amino acids 10–96 were compared in detail with the germline VH186.2 sequence as described (Smith et al., 2000).

Chemotaxis assay

Fo B cells purified by magnetic-activated cell sorting were activated with 5 µg/ml goat anti–mouse IgM (Jackson ImmunoResearch) for 24 h or rested overnight (unstimulated control). Various dilutions of recombinant mouse CCL21 (provided by the late I. Clark-Lewis) or CXCL13 (PeproTech) in 150 µl chemotaxis buffer (RPMI-1640 with 0.5% BSA and 20 mM Hepes) were added to the lower chambers of Transwell chemotaxis plates (96-well, 5-µm pore size; Corning). Cells were extensively washed in chemotaxis buffer and loaded into the upper chambers at 105 cells/well in 50 µl chemotaxis buffer and incubated for 3 h at 37°C. To enumerate B cell migration, cells were harvested from the bottom chambers, and B220+ cells were assessed by flow cytometry using a defined number of CaliBRITE beads (BD) as an internal reference. The migration index was calculated as described (Kara et al., 2013).

Statistics

Data were analyzed with Prism (GraphPad Software) using either two-tailed (unpaired or paired, as appropriate) Student’s t tests (for normally distributed data sets comparing the mean of two samples), two-tailed nonparametric Mann-Whitney tests (for data sets that were determined by an F test not to have a normal distribution and where there was a comparison of the mean of two samples), or one-way analysis of variance with appropriate posttests as indicated in text (for comparisons of multiple samples). For all analyses, P < 0.05 was considered significant. Sample or experiment sizes were determined empirically for sufficient statistical power. No statistical tests were used to predetermine the size of experiments. No data points were excluded from statistical tests.

Online supplemental material

Fig. S1 shows that follicular T cell responses to SRBC immunization are enhanced in the absence of hematopoietic or B cell expression of ACKR4. Fig. S2 shows the generation and characterization of Rosa26LSL-Ackr4 knockin mice.

Acknowledgments

We thank Josef Nguyen (Royal Adelaide Hospital, Adelaide, Australia) for mouse irradiation; the staff of Laboratory Animal Services, University of Adelaide, for animal husbandry; Meinrad Busslinger for Cd23Cre mice; and Harald Hartweger for comments on the manuscript.

This work was supported in part by a grant from the Australian National Health and Medical Research Council (APP1105312) to S.R. McColl, J.G. Cyster, and I. Comerford, J.G. Cyster is an investigator of the Howard Hughes Medical Institute. E.E. Kara is supported by an Australian postgraduate award, a Norman and Patricia Polglase scholarship, and a National Health and Medical Research Council C.J. Martin Overseas Biomedical fellowship.

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